Imaging agents for early detection and monitoring of cardiovascular plaque

ABSTRACT

The invention provides imaging agents comprising a label in association with a plaque specific targeting molecule. Methods for using the imaging agents to diagnose or monitor plaque formation and growth and kits containing the cardiovascular agents or components suitable for production of the imaging agents are also provided.

RELATED APPLICATIONS

This application is a 371 of Application No. PCT/US98/18685, filed Sep.8, 1998, which claims priority to U.S. application Ser. No. 08/925,213,filed Sep. 8, 1997, now abandoned.

The present invention is in the field of nuclear medicine. Morespecifically, the invention relates to imaging of plaque formation incardiovascular tissue.

BACKGROUND OF THE INVENTION

It is estimated that more than 1.5 million myocardial infarctions occurannually in the United States, and at least 500,000 infarctions resultin death, usually sudden. (American Heart Association, Heart and StrokeFacts. Dallas, Tex: American Heart Association National Center; 1992).Accordingly, myocardial infarction is the most frequent cause ofmortality in the United States; and in most Western countries (Coopers,ES. Prevention: The Key to Progress. Circulation. 1993; 24: 629–632;WHO-MONICA Project. Myocardial Infarction and Coronary Deaths in theWorld Health Organization Monica Project: Registration Procedures, EventRates and Care Fatality Rates in 38 Populations From 21 Countries inFour Continents. Circulation. 1994; 90:583–612). However, even theoptimal use of thrombolytic therapy for myocardial infarction, theadvance of which the greates attention has been focused, could preventonly 25,000 deaths or 5% of the total, because most deaths occursuddenly, before any type of treatment can be initiated. (Muller, J E,et al., Acute Risk Factors and Vulnerable Plaques: The Lexicon of a NewFrontier. J. Am. Coll. Cardiol. 1994; 23:809–813).

In 1992, Fuster et al., (Fuster V. et al., The Pathogenesis of CoronaryArtery Disease and the Acute Coronary Syndromes. N. Engl. J. Med. 1992;326:242–250.) classified the progression of coronary atheroscleroticdisease into five phases. Phase I is represented by a small plaque thatis present in most people under the age of 30 years regardless of theircountry of origin and that usually progresses slowly (types I to IIIlesions). Phase 2 is represented by a plaque, not necessarily verystenotic, with a high lipid content that is very prone to rupture (typesIV and Va lesions). The plaque of phase 23 may rupture withpredisposition to change its geometry and to formation of muralthrombus, these processes by definition represent phase 3 (type Ilesion), with a subsequent increase in stenosis, possibly resulting inangina, or ischemic sudden death. The mural and occlusive thrombi fromplaques of phases 3 and 4, by being organized by connective tissue, maycontribute to the progression of the atherosclerotic process representedby severely stenotic or occlusive plaques of phase 5 (types Vb and Vclesions). The severely stenotic plaques of phase 5, by a phenomenon ofstasis and/or deendothelialization, can become complicated by a thrombusand/or rapid myoproliferative response, also leading to an occlusiveplaque of phase 5. Of interest, about two thirds of coronary occlusionsare the result of this late stenotic type of plaque and are unrelated toplaque disruption. Unlike the rupture of less-stenotic lipid-richplaques, leading to occlusion and subsequent infarction or other acutecoronary syndromes, this process of occlusion from late stenotic plaquestends to be silent because the preceding severe stenosis and ischemiaenhance protective collateral circulation. (Fuster, V et al., ThePathogenesis of Coronary Artery Disease and the Acute CoronarySyndromes. N. Engl. J. Med. 1992; 326:242–250; Chesebro, J H et al.,Antithrombotic Therapy and Progression of Coronary Artery Disease.Circulation. 1992; 86 (suppl III)).

Sensitive and specific agents are needed to identify the early stages ofplaque formation in a subject, the progression of which can then bedelayed or reduced by initiation of an appropriate therapeutic regimenor change in lifestyle.

SUMMARY OF THE INVENTION

In general, the invention features imaging agents comprised of atargeting moiety and a label, such as a radionuclide or paramagneticcontrast agent. In preferred embodiments, the labeled imaging agentscomprise small molecule that rapidly (i.e. less than about 24 hours,more preferably less than about 12 hours and most preferably less thanabout 6 hours) localize, selectively and irreversibly localize at thesite of a plaque and rapidly clear from other tissue.

Examples of appropriate radionuclides include: ¹³¹I, ¹²⁵I, ¹²³I,^(99m)Tc, ¹⁸F, ⁶⁸Ga, ⁶⁷Ga, ⁷²As, ⁸⁹Zr, ⁶⁴Cu, ⁶²Cu, ¹¹¹In, ²⁰³Pb, ¹⁹⁸Hg,⁹⁷Ru, ¹¹C and ²⁰¹TI. Suitable paramagnetic contrast agents includegadolinium, cobalt, nickel, manganese and iron. Particularly preferredradionuclides or paramagnetic contrast agents have an appropriatehalf-life and high specific activity.

Particularly preferred targeting moieties comprise components of theprocesses involved in plaque formation and growth as well as specificbind partners thereto (e.g. receptors and fragments thereof, receptorligands, and antibodies and binding fragments thereof). Particularlypreferred targeting moieties are comprised of components of processesinvolved in plaque formation and growth as well as specific partner tosuch components (e.g. receptors and fragments thereof, receptor ligands(e.g receptor agonists or antagonists), and antibodies and bindingfragments thereof). Examples include: (i) cells, including smooth musclecells, leukocytes, lymphocytes and (B-lymophocytes), monocytes,macrophages, foam cells, platelets, erythrocytes and polymorphonuclearcells (e.g. granulocytes and neutrophils) and cellular fragments (e.g.heme) and analogs thereof (e.g. porphoryins and phthalocyanines); (ii)molecules that attract or modify cellular migration includingchemotactic proteins and peptides (e.g. monocyte chemotactic protein 1(MCP-1) and N-formyl-methionyl-leucyl-phenalanine other formyl peptides;colony stimulating factors (e.g. GM-CSF and CSF-1 and receptors andantibodies thereto; and platelet factor 4 (iii) growth factors (e.g.transforming growth factors, e.g. TGF-β, endothelial growth factors(e.g. VEGF) and growth factors that initiate smooth muscleproliferation), (iii) adhesive cell-surface glycoproteins (e.g.E-selectin, VCAM-1 and VCAM1β and; and carbohydrates such as¹¹C-deoxy-D-glucose and ¹⁸F-2-fluorodeoxy-D-glucose); (iv) othercomponents of a vascular inflammatory response (for examples complementcomponents (e.g. C1, C1q, C1r, C1s, C2, C3, C3a, C3b, C4, C4C2, C4C2C3b,C5a, C5b and C5a), immunoglobulins and cytokines (e.g. interleukins(e.g. IL-1, (IL-1α and IL-1β, IL-2; IL-3; IL-6; IL-7; and IL-8)interferons (interferon α, interferon γ) and tumor necrosis factors(e.g. TNF-α); (v) cellular sources of energy for metabolically activeplaque formation; (vi) lipids (e.g. liposomes, including polyethyleneglycol (PEG) coated liposomes, cholesterol and its esters, lipoproteins(e.g. LDL, HDL, oxidized LDL) and lipid receptors; and (vii) componentsof the clotting cascade (e.g. fibrin, thrombin, fibrinogen, factor VIII,factor IX, etc.)

In another aspect, the invention relates to methods for making theimaging agents. In a preferred embodiment, an appropriate label isionically or covalently associated with the targeting moity via any of avariety of means. In a preferred embodiment, the association is viaincorporation of a chelating structure, such as —N₂S₂, —NS₃, —N₄, anisonitrile, a hydrazine, a HYNIC (hydrazinonicotinic acid),2-methylthiolnicotinic acid, phosphorus, or a carboxylate containinggroup.

In yet another aspect, the invention features methods for imaging asubject for plaque formation and growth comprising administering to thesubject an effective amount of an imaging agent of the invention anddetecting the concentration and spatial distribution of the agent usingan appropriate detection means, wherein a higher differentialaccumulation of the agent in a particular location relative to otherlocations within the cardiovascular tissue of a subject is indicative ofplaque formation in the subject and wherein a higher differentialaccumulation of the agent in a particular location relative to theaccumulation detected at the same location in a prior imaging isindicative of plaque growth.

In yet a further aspect, the invention features a kit for imaging whichincludes, but is not limited to, a supply of the imaging agent or itsprecursor. The kit may also include at least one chelating structureand/or an auxiliary molecule such as, mannitol, gluconate,glucoheptonate, and tartrate; and a tin containing reducing agent.

Other features or advantages of the present invention will be apparentfrom the following detailed description and from the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

For convenience, the meaning of certain terms and phrases employed inthe following specification, examples and appended claims are providedbelow:

An “antibody or fragment thereof” refers to a whole polyclonal ormonoclonal antibody or a binding fragment therof.

A “chelating structure” refers to any molecule or complex of moleculeswhich bind to both the label and targeting moiety. Examples include:N₂S₂ structure, an NS₃ structure, an N₄ structure, anisonitrile-containing structure, a hydrazine containing structure, aHYNIC (hydrazinonicotinic acid) group-containing structure, a2-methylthiolnicotinic acid group-containing structure, a carboxylategroup containing structure, and the like.

“Cardiovascular disease” or “cardiovascular lesion” refers to any of avariety of disease or lesions to the heart or vasculature of a subject.Examples include atherosclerosis (i.e. thickening and hardening ofarteries due to plaque formation) and related disorders resulting fromoccluded blood flow (e.g. angina, cerebral ischemia, renal hypertension,ischemic heart disease, stroke) and thrombus and formation (e.g. DeepVein Thrombosis (DVT)).

“Cardiovascular tissue” refers to any and all tissue comprising thecardiovascular system including: all components of the heart, aortas,arteries (e.g. coronary and carotid), veins, or components of thesetissues and organs.

A “precursor of an imaging agent” refers to any molecule or complexes ofmolecules which are easily converted to the imaging agent.

A “small molecule” refers to a composition having a molecular weight,which is less than about 5KD, more preferably less than about 4KD, evenmore preferably less than about 3KD and most preferably less than about2 KD.

“Subject” refers to an animal, e.g. mammal, particularly a human.

A “targeting moiety or precursor thereof” is any molecule or biologicalentity that targets cardiovascular tissue or thrombi, or any molecule orbiological entity that is easily converted to such a molecule orbiological entity.

“thrombus” refers to a clot of blood formed within a blood vessel from aplaque and which remains attached to its place of origin.

“vascular inflammation” refers to vascular tissue damage in a subject,which may result from a number of causes (e.g. microbial infection,autoimmune processes, any injury or trauma, etc). Regardless of cause,the vascular inflammatory response consists of a complicated set offunctional and cellular adjustments involving changes inmicrocirculation, movement of fluids, proliferation of smooth musclecells, generation of foam cells and influx and activation ofinflammatory cells.

The present invention provides novel imaging agents which are comprisedof a targeting moiety and a label. These novel imaging agentsspecifically accumulate in actively forming or actively growing plaquesand therefore are useful for detecting or monitoring plaque formation.

Particularly preferred targeting moieties are comprised of components ofprocesses involved in plaque formation and growth as well as specificbind partners to such components (e.g. receptors and fragments thereof,receptor ligands (e.g receptor agonists or antagonists), and antibodiesand binding fragments thereof). Examples include: (i) cells, includingsmooth muscle cells, leukocytes, lymphocytes (B-lymphocytes andT-lymophocytes), monocytes, macrophages, foam cells, platelets,erythrocytes and polymorphonuclear cells (e.g. granulocytes andneutrophils) and cellular fragments and analogs thereof (e.g.porphoryins, such as heme and phthalocyanines); (ii) molecules thatattract or modify cellular migration including chemotactic proteins andpeptides (e.g. monocyte chemotactic protein 1 (MCP-1) andN-formyl-methionyl-leucyl-phenalanine (See U.S. Pat. No. 5,792,444)other formyl peptides; colony stimulating factors (e.g. GM-CSF (See U.S.Pat. Nos. 5,229,496 and 4,879,227) and CSF-1 (See U.S. Pat. Nos.4,847,201; 4,868,119 and 4,929,700 and receptors and antibodies thereto;and platelet factor 4 (iii) growth factors (e.g. transforming growthfactors, e.g. TGF-β, endothelial growth factors (e.g. VEGF) and growthfactors that initiate smooth muscle proliferation), (iii) adhesivecell-surface glycoproteins (e.g. E-selectin, VCAM-1 and VCAM1β (See e.g.U.S. Pat. No. 5,272,263) and ICAM-1 (See Rosenfeld, M E et al.,Cellularity of Atherosclerotic Lesions Car. Art. Dis. 1994; 5:189–197;Navab, M. et al., Monocyte Adhesion and Transmigration inAtherosclerosis. Cor Art. Dis. 1994: 5: 198–204) and other cell bindingmolecules See e.g Kim, J A et al., Partial Characterization of LeukocyteBinding Molecules on Endothelial Cells Induced by Minimally Oxidized LDLArterio. Thromb. 1994; 24: 427–433)); and carbohydrates such as¹¹C-deoxy-D-glucose and ¹⁸F-2-fluorodeoxy-D-glucose); (iv) othercomponents of a vascular inflammatory response (for examples complementcomponents (e.g. C1, C1q, C1r, C1s, C2, C3, C3a, C3b, C4, C4C2, C4C2C3b,C5a, C5b and C5a), immunoglobulins and cytokines (e.g. interleukins(e.g. IL-1, (IL-1α (See U.S. Pat. No. 4,762,914) and IL-1β (See U.S.Pat. No. 4,766,061), IL-2 (See U.S. Pat. Nos. 5,037,644; 4,939,093;4,604,377; and 4,518,584); IL-3; IL-4 (See U.S. Pat. No. 5,017,691);IL-6; IL-7; and IL-8) interferons (interferon α, interferon γ) and tumornecrosis factors (e.g. TNF-α); (v) cellular sources of energy formetabolically active plaque formation; (vi) lipids (e.g. liposomes,including polyethylene glycol (PEG) coated liposomes, cholesterol andits esters, lipoproteins (e.g. LDL, HDL, oxidized LDL) and lipidreceptors; and (vii) components of the clotting cascade (e.g. fibrin,thrombin, fibrinogen, factor VIII, factor IX, etc.)

In accordance with the invention, the targeting molecule is inassociation with (spatial proximity to) the label. Spatial proximitybetween the targeting molecule and the label may be effected in anymanner which preserves the specificity of the targeting molecule for itstarget tissue. For example, spatial proximity between the label and thetargeting molecule may be effected by a covalent or non-covalentchemical bond. Such a chemical bond may be effected through a chelatingsubstance and/or an auxiliary molecule such as mannitol, gluconate,glucoheptonate, tartrate, and the like. Alternatively, spatial proximitybetween the label and the targeting molecule may be effected byincorporating the label and the targeting molecule in a micelle orliposome, in such a way that the affinity of the targeting molecule forits target tissue is maintained. Spatial proximity between the label andthe targeting molecule may also be effected by attaching the label andthe targeting molecule to a matrix such as a microsphere, liposome, ormicelle.

The imaging agents described above may contain any label in accordancewith the invention. Highly specific and sensitive labels are provided byradionuclides, which can then be detected, using positron emissiontomography (PET) or Single Photon Emission Computed Tomography (SPECT)imaging. More preferably, the imaging agent of the invention contains aradionuclide selected from the group consisting of ¹³¹I, ¹²⁵I, ¹²³I,^(99m)Tc, ¹⁸F, ⁶⁸Ga, ⁶⁷Ga, ⁷²As, ⁸⁹Zr, ⁶⁴Cu ⁶²Cu, ¹¹¹In, ²⁰³Pb, ¹⁹⁸Hg,¹¹C, ⁹⁷Ru, and ²⁰¹TI or a paramagnetic contrast agent, such asgadolinium, cobalt, nickel, manganese and iron. Such labels may beincorporated into the imaging agent by covalent bonding directly to anatom of the targeting molecule, or the label may be non-covalently orcovalently associated with the targeting molecule through a chelatingstructure or through an auxiliary molecule such as mannitol, gluconate,glucoheptonate, tartrate, and the like. When a chelating structure isused to provide spatial proximity between the label and the targetingmolecule, the chelating structure may be directly associated with thetargeting molecule or it may be associated with the targeting moleculethrough an auxiliary molecule such as mannitol, gluconate,glucoheptonate, tartrate, and the like.

Any suitable chelating structure may be used to provide spatialproximity between the radionuclide and the targeting molecule of theagent through covalent or noncovalent association. Many such chelatingstructures are known in the art. Preferably, the chelating structure isan N₂S₂ structure, an NS₃ structure, an N₄ structure, anisonitrile-containing structure, a hydrazine containing structure, aHYNIC (hydrazinonicotinic acid) group-containing structure, a2-methylthiolnicotinic acid group-containing structure, a carboxylategroup containing structure, and the like. In some cases, chelation canbe achieved without including a separate chelating structure, becausethe radionuclide chelates directly to atom(s) in the targeting moiety,for example to oxygen atoms in various moieties.

The chelating structure, auxiliary molecule, or radionuclide may beplaced in spatial proximity to any position of the targeting moleculewhich does not interfere with the interaction of the targeting moleculewith its target site in cardiovascular tissue. Accordingly, thechelating structure, auxiliary molecule, or radionuclide may becovalently or non-covalently associated with any moiety of the targetingmolecule except the receptor-binding moiety.

Radionuclides may be placed in spatial proximity to the targetingmolecule using known procedures which effect or optimize chelation,association, or attachment of the specific radionuclide to ligands. Forexample, when ¹²³I is the radionuclide, the imaging agent may be labeledin accordance with the known radioiodination procedures such as directradioiodination with chloramine T, radioiodination exchange for ahalogen or an organometallic group, and the like. When the radionuclideis ^(99m)Tc, the imaging agent may be labeled using any method suitablefor attaching ^(99m)Tc to a ligand molecule. Preferably, when theradionuclide is ^(99m)Tc, an auxiliary molecule such as mannitol,gluconate, glucoheptonate, or tartrate is included in the labelingreaction mixture, with or without a chelating structure. Morepreferably, ^(99m)Tc is placed in spatial proximity to the targetingmolecule by reducing ^(99m)TcO₄ with tin in the presence of mannitol andthe targeting molecule. Other reducing agents, including tin tartrate ornon-tin reductants such as sodium dithionite, may also be used to makethe cardiovascular imaging agent of the invention.

In general, labeling methodologies vary with the choice of radionuclide,the moiety to be labeled and the clinical condition under investigation.Labeling methods using ^(99m)Tc and ¹¹¹In are described for example inPeters, A. M. et al., Lancet 2: 946–949 (1986); Srivastava, S. C. etal., Semin. Nucl. Med. 14(2):68–82 (1984); Sinn, H. et al., Nucl. Med.(Stuttgart) 13:180, 1984); McAfee, J. G. et al., J. Nucl. Med.17:480–487, 1976; McAfee, J. G. et al., J. Nucl. Med. 17:480–487, 1976;Welch, M. J. et al., J. Nucl. Med. 18:558–562, 1977; McAfee, J. G., etal., Semin. Nucl. Med. 14(2):83, 1984; Thakur, M. L., et al., Semin.Nucl. Med. 14(2):107, 1984; Danpure, H. J. et al., Br. J. Radiol.,54:597–601, 1981; Danpure, H. J. et al., Br. J. Radiol. 55:247–249,1982; Peters, A. M. et al., J. Nucl. Med. 24:39–44, 1982; Gunter, K. P.et al., Radiology 149:563–566, 1983; and Thakur, M. L. et al., J. Nucl.Med. 26:518–523, 1985.

After the labeling reaction is complete, the reaction mixture mayoptionally be purified using one or more chromatography steps such asSep Pack or high performance liquid chromatography (HPLC). Any suitableHPLC system may be used if a purification step is performed, and theyield of cardiovascular imaging agent obtained from the HPLC step may beoptimized by varying the parameters of the HPLC system, as is known inthe art. Any HPLC parameter may be varied to optimize the yield of thecardiovascular imaging agent of the invention. For example, the Ph maybe varied, e.g., raised to decrease the elution time of the peakcorresponding to the cardiovascular imaging agent of the invention.

The invention as embodied in a kit for imaging comprises one or more ofthe imaging agents described above, in combination with apharmaceutically acceptable carrier such as human serum albumin. Humanserum albumin for use in the kit of the invention may be made in anyway, for example, through purification of the protein from human serumor though recombinant expression of a vector containing a gene encodinghuman serum albumin. Other substances may also be used as carriers inaccordance with this embodiment of the invention, for example,detergents, dilute alcohols, carbohydrates, auxiliary molecules, and thelike. The kit of the invention may of course also contain such otheritems as may facilitate its use, such as syringes, instructions,reaction vials, and the like.

In one embodiment, a kit according to the invention contains from about1 to about 30 mCi of the radionuclide-labeled cardiovascular imagingagent described above, in combination with a pharmaceutically acceptablecarrier. The cardiovascular imaging agent and carrier may be provided insolution or in lyophilized form. When the cardiovascular imaging agentand carrier of the kit are in lyophilized form, the kit may optionallycontain a sterile and physiologically acceptable reconstitution mediumsuch as water, saline, buffered saline, and the like.

In another embodiment, the kit of the invention may contain theunlabeled targeting molecule which has been covalently or non-covalentlycombined with a chelating agent; an auxiliary molecule such as mannitol,gluconate, glucoheptonate, tartrate, and the like; and a reducing agentsuch as SnCl₂ or tin tartrate. The unlabeled targetingmolecule/chelating agent and the auxiliary molecule may be present asseparate components of the kit or they may be combined into one kitcomponent. The unlabeled targeting molecule/chelating agent, theauxiliary molecule, and the reducing agent may be provided in solutionor in lyophilized form, and these components of the kit of the inventionmay optionally contain stabilizers such as NaCl, silicate, phosphatebuffers, ascorbic acid, gentisic acid, and the like. Additionalstabilization of kit components may be provided in this embodiment, forexample, by providing the reducing agent in an oxidation-resistant form.

Determination and optimization of such stabilizers and stabilizationmethods are well within the level of skill in the art. When theunlabeled targeting molecule/chelating, agent of this embodiment are inlyophilized form, the kit may optionally contain a sterile andphysiologically acceptable reconstitution medium such as water, saline,buffered saline, and the like. The amounts of unlabeled targetingmolecule/chelating agent, auxiliary molecule, and reducing agent in thisembodiment can be optimized in accordance with the methods for makingthe cardiovascular imaging agent set forth above. Radionuclides,including, but not limited to, ^(99m)Tc, e.g. obtained from acommercially available ⁹⁹Mo/^(99m)Tc generator or commercially available¹²³I, may be combined with the unlabeled targeting molecule/chelatingagent and the reducing agent for a sufficient period of time and at atemperature sufficient to chelate the radionuclide to the targetingmolecule/chelating agent, and the imaging agent thus formed is injectedinto the patient.

The cardiovascular imaging agents of the invention may be used inaccordance with the methods of the invention by those of skill in theart, e.g., by specialists in nuclear medicine, to image plaque in thecardiovascular system of a subject. Images are generated by virtue ofdifferences in the spatial distribution of the imaging agents whichaccumulate in the various tissues and organs of the subject. The spatialdistribution of the imaging agent accumulated may be measured using anysuitable means, for example, a gamma camera, a PET apparatus, a SPECTapparatus, and the like. Some cardiovascular lesions may be evident whena less intense spot appears within the image, indicating the presence oftissue in which a lower concentration of imaging agent accumulatesrelative to the concentration of imaging agent which accumulates insurrounding cardiovascular tissue. Alternatively, a cardiovascularlesion might be detectable as a more intense spot within the image,indicating a region of enhanced concentration of the imaging agent atthe site of the lesion relative to the concentration of agent whichaccumulates in surrounding cardiovascular tissue. Thrombi and embolismsare examples of cardiovascular lesions which accumulate enhancedconcentrations of the imaging agents of the invention. Accumulation oflower or higher amounts of the imaging at the site of a lesion mayreadily be detected visually, by inspection of the image of thecardiovascular tissue. Alternatively, the extent of accumulation of theimaging agent may be quantified using known methods for quantifyingradioactive emissions. A particularly useful imaging approach employsmore than one imaging agent to perform simultaneous studies. Forexample, simultaneous studies of perfusion and metabolic function wouldallow study of coupling and uncoupling of flow of metabolism, thusfacilitating determinations of tissue viability after a cardiac injury.Such determinations are useful in diagnosis of cardiac ischemia,cardiomyopathy, tissue viability, hibernating heart, and other heartabnormalities.

An effective amount of an imaging agent comprising at least onetargeting molecule and a label (e.g. from about 1 to about 50 mCi of aradionuclide) may be combined with a pharmaceutically acceptable carrierfor use in imaging studies. In accordance with the invention, “aneffective amount” of the imaging agent of the invention is defined as anamount sufficient to yield an acceptable image using equipment which isavailable for clinical use. An effective amount of the imaging agent ofthe invention may be administered in more than one injection. Effectiveamounts of the imaging agent of the invention will vary according tofactors such as the degree of susceptibility of the individual, the age,sex, and weight of the individual, idiosyncratic responses of theindividual, the dosimetry. Effective amounts of the imaging agent of theinvention will also vary according to instrument and film-relatedfactors. Optimization of such factors is well within the level of skillin the art. In general, the effective amount will be in the range offrom about 0.1 to about 10 mg by injection or from about 5 to about 100mg. orally for use with MRI.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic agents, absorption delaying agents, and the like. Theformulation used in the present invention may also contain stabilizers,preservatives, buffers, antioxidants, or other additives known to thoseof skill in the art. The use of such media and agents forpharmaceutically active substances is well known in the art.Supplementary active compounds can also be incorporated into the imagingagent or the invention. The imaging agent of the invention may furtherbe administered to an individual in an appropriate diluent or adjuvant,co-administered with enzyme inhibitors or in an appropriate carrier suchas human serum albumin or liposomes. Pharmaceutically acceptablediluents include sterile saline and other aqueous buffer solutions.Adjuvants contemplated herein include resorcinols, non-ionic surfactantssuch as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.Enzyme inhibitors include pancreatic trypsin inhibitor,diethylpyrocarbonate, and trasylol. Liposomes inhibitors includewater-in-oil-in-water CGF emulsions as well as conventional liposomes(Strejan et al., J Neuroimmunol 7:27 [1984]).

The subject imaging agents can be administered to a subject inaccordance with any means that facilitates accumulation of the agent ina subject's cardiovascular system. Preferably, the imaging agent of theinvention is administered by arterial or venous injection, and has beenformulated as a sterile, pyrogen-free, parenterally acceptable aqueoussolution. The preparation of such parenterally acceptable solutions,having due regard to pH, isotonicity, stability, and the like, is withinthe skill in the art. A preferred formulation for intravenous injectionshould contain, in addition to the cardiovascular imaging agent, anisotonic vehicle such as Sodium Chloride Injection, Ringer's Injection,Dextrose Injection, Dextrose and Sodium Chloride Injection, LactatedRinger's Injection, or other vehicle as known in the art.

The amount of imaging agent used for diagnostic purposes and theduration of the imaging study will depend upon the nature and severityof the condition being treated, on the nature of therapeutic treatmentswhich the patient has undergone, and on the idiosyncratic responses ofthe patient. Ultimately, the attending physician will decide the amountof imaging agent to administer to each individual patient and theduration of the imaging study.

The present invention is further illustrated by the following examples,which should not be construed as limiting in any way. The contents ofall cited references (including literature references, issued patents,published patent applications) as cited throughout this application arehereby expressly incorporated by reference.

EXAMPLE Preparation of Radiolabeled Chemotactic Peptide For-MLF

For-MLF is a bacterial product that initiates leukocyte chemotaxis bybinding to high affinity receptors on white blood cell membranes(Showell et al., J Exp Med 143:1154–1169 [1976], Schiffmann et al., ProcNatl Acad Sci USA 72:1059–1062 [1975], Williams et al., Proc Natl AcadSci 74:1204–1208 [1977]). These receptors are present on bothpolymorphonuclear leukocytes and mononuclear phagocytes. Due to the verysmall size of For-MLF (MW 437), its molecular structure can be readilymanipulated to design an optimal imaging agent.

The labelled chemotactic peptide can be synthesized and purified by thetechniques described in Babich et al., J Nucl Med 34:1964–1974 (1993).

Dimethylformamide (DMF) (2 ml) and 60 μl of diisopropylethylamine isadded to 186 mg of N-For-Met-Leu-Phe-diaminohexyl amide followed by 154mg succinimidyl-6-t-BOC-hydrazinopyridine-3-carboxylic acid in 1 ml DMF.The mixture becomes yellow and the peptide dissolves within a shorttime. After 2 hours, ether is added to the reaction mixture and theupper layer is discarded. Water is added to the oily residue causing asolid to form. The solid is washed with 5% sodium bicarbonate, water andethyl acetate, and the yield is determined. The t-BOC protecting groupis removed by stirring the crude product with 5 ml of trifluoroaceticacid (TFA) containing 0.1 ml of p-cresol for 15 min. at 20° C. Prolongedtreatment with TFA results in increased levels of a side product. TheTFA is removed by rotary evaporation, and ether is added to the residueto precipitate the deprotected peptide. The product is purified byreverse phase HPLC on a 2.5×50 cm Whatman ODS-3 column is eluted with agradient of acetonitrile in 0.1% TFA. Fractions containing the majorcomponent is combined and the solvent is removed to yield the desiredproduct.

Technetium-99m-pertechnetate (⁹⁹Mo/^(99m)Tc generator) and stannousglucoheptonate (Glucoscan) are obtained from New England Nuclear(Boston, Mass.). Technetium-99m-glucoheptonate is used to provide thenecessary Tc(V) oxo species for radiolabeling the hydrazinonicotinamideconjugated peptides. Approximately 2.S ml of ^(99m)Tc-pertechnetate in0.9% of NaCl is added to the freeze-dried kit. The final radioactiveconcentration is 5–10 mCi/ml and radiochemical purity of the product isdetermined by instant thin-layer silica gel chromatography (ITLC-sg)using both acetone and 0.9% NaCl as mobile phase solvents.

Approximately 0.2 mg of peptide is dissolved in 50 μl dimethylsulfoxideand the solution is diluted to a final concentration of 0.1 mg/ml with0.1 M acetate buffer pH 5.2. Peptide solution (0.5 ml) is placed in aclean glass vial and 0.5 ml of ^(99m)Tc-glucoheptonate is added. Themixture is vortexed briefly and is allowed to stand at room temperaturefor 1 hour. Radiochemical purity is determined by ITLC-sg in threesolvent systems: acetone, 0.9% NaCl, and acetone and water (9:1).

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A cardiovascular imaging agent comprising a radionuclide, said radionuclide being chemically bonded to a targeting moiety comprising a component of a process involved in plaque formation, wherein the targeting moiety is fibrin, thrombin, fibrinogen, factor VIII, or factor IX, wherein said radionuclide is a positron emitting radionuclide selected from the following: ¹⁸F, ⁶⁸Ga, ⁶²Cu, or radioactive isotopes of iodine.
 2. The agent of claim 1, wherein said plaque is an atherosclerotic forming plaque.
 3. A method of imaging cardiovascular plaque formation in a mammal, comprising administering to the mammal a cardiovascular imaging agent having a radionuclide, said radionuclide being chemically bonded to a targeting moiety comprising a component of a process involved in plaque formation, wherein the targeting moiety is fibrin, thrombin, fibrinogen, factor VIII, or factor IX, wherein said radionuclide is a positron emitting radionuclide selected from the following: ¹⁸F, ⁶⁸Ga, ⁶²Cu, or radioactive isotopes of iodine.
 4. The method of claim 3, wherein the method detects a cardiovascular lesion in a mammal, said method comprising the steps of administering to the mammal said imaging agent, detecting the spatial distribution of said agent accumulated in the mammal's cardiovascular system, wherein a detected accumulation of said agent in a region which is different from the detected accumulation of said agent in other regions is indicative of a lesion.
 5. The method of claim 4, wherein said cardiovascular lesion is an atherosclerotic forming lesion.
 6. A kit for cardiovascular imaging, comprising a supply of the imaging agent or a precursor of the imaging agent having a radionuclide, said radionuclide being chemically bonded to a targeting moiety comprising a component of a process involved in plaque formation, wherein the targeting moiety is fibrin, thrombin, fibrinogen, factor VIII, or factor IX, wherein said radionuclide is a positron emitting radionuclide selected from the following: ¹⁸F, ⁶⁸Ga, ⁶²Cu, or radioactive isotopes of iodine. 